Lung immaturity leads to decreased pulmonary compliance and respiratory distress. The mainstay in management is oxygen and respiratory support. However, these therapies can cause oxidative injury and lead to further pulmonary disease. During oxidant therapy, antioxidant systems are critical in reducing the burden of toxic oxygen intermediates and preventing tissue injury. In premature infants, injury from reactive oxygen species generated during oxygen therapy is compounded by a poorly developed antioxidant system. Glutathione (GSH) is an essential component of the pulmonary antioxidant system and premature birth is associated with decreased GSH in lung tissue and in the fluid lining the alveolar surface. The decrease in GSH of the epithelial lining fluid and the injury were inversely related to the gestational age and suggest the importance of the GSH content of the epithelial lining fluid. This decrease in the epithelial lining fluid GSH could be due to plasma GSH availability, uptake by microvascular endothelial cells (MVEC), uptake by alveolar epithelial cells (AEC) and release of GSH onto the alveolar surface. With over 40 different cell types in the lung, studies of GSH transport with in vivo and whole organ models are difficult to interpret. Studies of cultured MVEC or cultured AEC from fetal animals will provide important insight into the development of GSH transport and the impact of hyperoxic exposure on that transport. However, studies of either cell type in isolation will provide only partial insight. A more complete picture of GSH transport would be obtained with a model where both MVED and AEC were present in the appropriate orientation. Using a CoStar Transwell, we have developed a bilayer model using primary MVEC and AEC from adult guinea pigs that are in a configuration where the MVEC basolateral surface in adjacent to the AEC basolateral surface. The AEC surface is an air interface rather than a liquid interface. The goal of this proposal is to develop a fetal bilayer with primary MVEC and AEC isolated from guinea pigs at different gestational ages (Aim 1). This fetal bilayer will then be used study gestational development of GSH transport from the endothelial surface to the epithelial surface (Aim 2) and the impact of hyperoxia on that transport process (Aim 3). The development of this fetal bilayer will be a useful model to study many physiological processes that require endothelial and epithelial cell interaction including neutrophil adhesion and migration.